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44839-B4
Epihalohydrin Cross-Linking of DNA
The goal of this project is to characterize DNA damage by the epihalohydrins. In the past year, we have made progress in the following areas:
1. We have published the sequence preference and stereospecificity for epihalohydrin cross-linking within synthetic DNA oligomers.
2. We are assessing epihalohydrin interstrand cross-links for bending.
3. We are determining the partitioning of epihalohydrins within different regions of DNA in a chicken erythroid cell line.
Goal 1 Accomplishments
We have been investigating the structure-function relationship for epichlorohydrin (ECH) and epibromohydrin (EBH). ECH is a common industrial cross-linking agent, but there were no prior literature reports that it cross-links DNA. We recently published the first report of interstrand cross-linking by both agents, finding that EBH is a more efficient cross-linker than ECH. The optimal pH for cross-linking also varies, with ECH more efficient at pH 5.0 and EBH more efficient at pH 7.0. Both agents are relatively flexible in the sequences targeted, with comparable efficiencies for 5'-GGC and 5'GC sites. We also found that R-ECH is both twice as effective at cross-linking as S-ECH and twice as cytotoxic, suggesting a correlation between the two activities.
Goal 2 Accomplishments
We are using native gel electrophoresis to examine potential DNA bending by cross-linkers such as the epihalohydrins. Cross-linked duplexes and control native duplexes are ligated to form a population of multimers that appear as a ladder of bands on a native gel. If the parent cross-linked duplex is bent, then this ladder of bands will appear retarded relative to a control lane of size standards of equal lengths. The magnitude of the anomalous mobility can be defined in terms of the ratio of the apparent length to the actual length in base pairs and can be used to calculate the angle of absolute curvature. We have found that long duplexes are cross-linked more efficiently than short ones, so we are currently performing these studies with duplexes of 30-40 base pairs in length. We have determined about a 23° bend per helical turn upon EBH cross-linking of a 30-mer and are attempting to verify this value in other duplexes.
Goal 3 Accomplishments
We have developed protocols to monitor the reactivity of ECH and related compounds within a 6C2 chicken erythroid cell line. We have assayed for damage within three different regions of chicken erythroid cells: mitochondrial DNA, expressed nuclear DNA, and unexpressed nuclear DNA. We have assessed the degree of damage at each site via a quantitative polymerase chain reaction (QPCR), which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We have found that ECH and EBH preferentially target nuclear DNA relative to mitochondrial DNA. Formation of lesions occurs at comparable levels for the nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation. Future plans include expanding the QPCR work into human cell lines.
Impact on Students
The Department of Chemistry at Colby College is firmly committed to providing significant independent research opportunities for all our students, and we therefore require independent research of our majors. We believe that independent research reinforces classroom instruction, teaches new techniques, and requires students to develop time-management skills, perseverance, and the ability to work in a group setting. Several students have been involved in this work in the Millard laboratory over the past year. Adam Newman '07 is beginning a Ph.D. program in chemical biology at Johns Hopkins University, Megan Watts '08 and Erin McGowan '08 are applying to M.D. and D.V.M. programs, respectively, and Sharonda Bradley '10 is entering her sophomore year at Colby College. Mr. Newman, Ms. Watts, and Ms. McGowan all presented their work at the national ACS meeting last spring. Mr. Newman, Ms. Watts, and Ms. Bradley spent the summer in the Millard lab, joining 16 other students in the Chemistry Department. The camaraderie among the students was tremendous, with the younger students bouncing ideas off the more experienced ones and gaining confidence in their own abilities. The following presentations have resulted from this work:
Quantitative PCR suggests preferential nuclear DNA alkylation by epichlorohydrin in the chicken genome (2007) Adam G. Newman, Frederick J. LaRiviere, and Julie T. Millard; Poster presented at the 233rd National Meeting of the American Chemical Society in Chicago IL.
Quantitative PCR methods for assessing epichlorohydrin damage within chicken erythroid cells (2007) Megan L. Watts and Julie T. Millard; Poster presented at the 233rd National Meeting of the American Chemical Society in Chicago IL.
Native polyacrylamide gel electrophoresis assessment of DNA bending upon diepoxybutane and epihalohydrin cross-linking (2007) Erin McGowan and Julie T. Millard; Poster presented at the 233rd National Meeting of the American Chemical Society in Chicago IL.
